For infants below the age of 24 months, the disease caused by Bordatella pertussis, pertussis or whooping cough, can be very severe and has a mortality rate of approximately 1%. Over the last fifty years, three types of vaccine have been available for immunization against the disease. The most widely used vaccine, that has been available for the protection of infants against pertussis infection, in combination with tetanus and diphtheria vaccines, is the so-called "whole cell" vaccine, which is available in all developed countries, and many of the Third World countries.
This whole cell pertussis vaccine is prepared by growing known strains of the B.pertussis organism in a defined medium for several hours in a fermentor, until the mixture reaches certain defined parameters. The mixture then is treated with a chemical agent, such as formaldehyde, which kills the organism and detoxifies proteins present in the supernatant and in the organism itself. After allowing the mixture to stand for a specified time to ensure that this detoxification procedure is complete, the cells are separated from the supernatant by passing the mixture through a continuous centrifuge, to provide a packed mass of cells and the supernatant, which is discarded. The cells then are resuspended in a solution of sodium chloride to provide a suspension, that, when diluted to a known strength, usually determined by the opacity of the suspension, and injected subcutaneously, elicits antibodies that are protective against the disease. This "whole cell" vaccine is known for giving minor local reactions at the injection site with occasionally more severe overall reactions, such as elevated temperature and general fretfulness. There has been speculation that the vaccine is responsible for some neurological reactions in infants.
In a second vaccine type, the B.pertussis was grown and detoxified with formaldehyde as before, and then the isolated cells extracted with a concentrated solution of urea. After filtration and dialysis to remove the urea, a mixture of soluble cell wall components is obtained, which, after dilution to a known strength, was in use as a vaccine from 1969 to 1974, but has now been withdrawn because of poor efficacy. More recently, a third vaccine type, commonly called an acellular vaccine, that is in use in Japan and is in clinical trials in a number of other developed countries, is prepared by isolation and purification of constituents of the culture supernatants of B.pertussis after detoxification. Specifically, the constituents called lymphocytosis promoting factor (LPF) also known as pertussis toxin (PT), filamentous hemaglutinin (FHA) and agglutinogens have been isolated and identified. However, because of variations in growth of the organism and the method of isolation, which is non-specific, the composition of the isolated mixture of proteins can vary. At present there is no vaccine in general use and licensed that is completely non-toxic and still gives good protection.
With the growth of the science of immunology over the years, it has been recognized that protective antibodies against a specific disease can be elicited by the administration of specific cellular components of the organism that causes the disease, rather than the whole organism that has been inactivated or has been attenuated to give a non-pathogenic strain. It has been recognized that use of detoxified or attenuated organisms as a vaccine can introduce components that may be damaging to the recipient. With this in mind, a number of efforts have been made to isolate components of the pertussis organism, either from the cell or that have been excreted into the medium, that could have antigenic capabilities, be completely non-toxic and thus could act as vaccines.
As yet there has been no convincing proof that any one particular cell component, by itself, can act as a protective antigen. However, in a number of publications and Patent applications (see, for instance, European Patent Application Nos. 0231083 and 0175841) it has been suggested that a mixture of the purified and detoxified proteins, lymphocytosis promoting factor (LPF) and filamentous hemaglutinin (FHA), can act as a combined antigen and, when administered to a mammal, generate antibodies that confer protection against the disease. In the aforesaid publications various methods have been disclosed for obtaining these proteins in a purified form, and once purified using them in varying proportions as a vaccine.
Although existing technology produces highly purified LPF and FHA, the processes have inherent drawbacks. The affinity chromatographic methods that have been used are effective but conditions of absorption and elution often use materials which are toxic, expensive and/or denature the required proteins. In addition, some of the materials used on affinity columns can be leached into the product under the harsh elution conditions required and, since some of the leached materials are blood derived, may introduce the possibility of blood born diseases or autosensitization. The use of gel filtration materials and hydroxyapatite are acceptable when used by themselves, but give only low purification factors.
The LPF and FHA, which are produced by B.pertussis, also represent a major challenge in the removal of lipopolysaccharides (LPS) as a contaminant. LPS even in nanogram quantities can produce fever and are an undesirable component of any vaccine. The initial concentration of LPS in the fermentation supernatant can be as high as one milligram per milliliter. A number of methods have been used to remove pyrogens from vaccines (see, for instance, U.S. Pat. Nos. 4,000,257 and 4,380,511). Many of these methods are too harsh and result in denaturation of the required proteins. Other methods are ineffective, cumbersome to use and expensive.
Before using the LPF and FHA in a vaccine, these proteins must be detoxified since the LPF in its natural state is highly toxic and small amounts are still present in the purified FHA. This process previously has been achieved by treating the proteins with a chemical agent which induces cross-linking. Traditionally, the agents used have been formaldehyde and glutaraldehyde. The use of formaldehyde and glutaraldehyde can lead to heavy losses due to aggregation and precipitation.